Cervical cancer remains one of the most common cancer types affecting women worldwide. The biological pathway to cervical carcinoma begins with normal intraepithelial cells, and develops through low and then high grade dysplasia before malignancy obtains. Cytologists mark the passage to malignancy as progression from normal epithelial cells to atypical squamous cells of undetermined significance (ASCUS) to Low Grade squamous intraepithelial lesions (LSIL) and then high grade squamous intraepithelial lesions (HSIL) before carcinoma in situ and finally malignancy result. Histologists mark the progression from normal cells to various grades of cervical intraepithelial neoplasia (CIN I, II and III), then to carcinoma in situ and finally malignancy. CIN I is considered low grade dysplasia comparable to LSIL. CIN II and III are considered high grade dysplasia comparable to HSIL.
The current standard of care includes regular cytologic testing with a Papanicolau (Pap) smear to identify abnormalities as indicating dysplasia or carcinoma in patient cells. When high grade dysplasia is detected and confirmed by histological examination, the transformation zone of the patient's cervix is removed immediately by loop excision or cone biopsy. More radical procedures are required when carcinoma is detected. At the same time, however, the progression from normal to malignancy is not strict and the presence of low grade dysplasia does not necessarily indicate that the patient will progress to high grade dysplasia or malignancy. Significantly, the negative predictive value of cytologic methods (e.g., Pap smears) for detecting high grade dysplasia is poor. Thus, low grade dysplasia may be misdiagnosed as high grade, thereby subjecting the patient to unwarranted treatment and high grade dysplasia may be misdiagnosed as low grade dysplasia, thereby delaying appropriate treatment. Accordingly, there is a need for a diagnostic method that will accurately distinguish between low and high grade dysplasia.
Patient specimens typically comprise many thousands of cells for evaluation. Diagnosis based on evaluation of individual cells can be enormously time consuming and tedious for technicians to perform due to the large number of cells that are required for evaluation. Thus, there is a need for a means to simplify a cell evaluation method.
Others have noted that genetic abnormalities (e.g., changes in chromosome regions or changes in ploidy levels) accompany the progression from normal cells to cervical malignancy. See, e.g., U.S. Pat. No. 5,919,624 to Ried, et al. Ried et al. noted that chromosomal abnormalities can be used to classify the progression of dysplastic cervical cells in late stages, e.g., from noninvasive cervical to invasive cervical carcinoma. Still others have demonstrated that cervical cancer is associated with infection by certain human papilloma viruses (HPV) types, particularly HPV types 16, 18, 31, 33, 35 and 42. See, e.g., Lazo, Brit. J. Cancer, (1999) 80(12), 2008-2018. Additionally, many cell cycle proteins such as p16 and Cyclin E and cell proliferation markers such as the proteins Ki67 and PCNA are also known to be highly active in neoplastic cells. Thus, cells containing abnormal amounts of these markers have been suggested as good candidates for cells that may progress to malignancy.
PCT application WO 0024760 describes methods and reagents for detecting HPV DNA in Pap smears using in situ hybridization and brightfield microscopy. The probe consists of full length DNA probes of HPV-16, -18, -31, -33, -35, and -51. The patent claims that this probe mix detects other high-risk HPV types but not low-risk HPV. The ability of the disclosed HPV probe mixture to avoid hybridization to low-risk HPV types is achieved by modulation of the quantities of each HPV DNA probe included on the probe mix. The HPV probes disclosed are different than those described herein. In addition, the assays of the invention modulate probe cross-hybridization by lowering the stringency of the hybridization conditions while keeping the probe concentrations constant for all types. This application also does not combine HPV probe with use of chromosomal probes to detect chromosome abnormalities in the HPV infected cells.
Hopman et al. (J of Pathology 2004; 202:23-33) analyzed HPV status and chromosomal aberrations in cervical biopsies sections by FISH. This work used only probes for HPV-16 and HPV-18 and genomic probes for chromosome 1 (1q12), 17, and X. In contrast to the inventive assay that simultaneously detect HPV and chromosomal gains in the same cells, Hopman et al.'s detection of HPV positive cells and chromosomal aberrations was performed in parallel tissue sections.
To date Applicants are not aware of any publication that has demonstrated that any chromosomal abnormality with or without the presence of another marker can be used to distinguish low from high grade dysplasia or has combined such a diagnostic method with the known association of HPV and cervical cancer.